1. Field of the Invention
This invention relates to mammalian blood plasma. More especially, this invention relates to the inactivation of hepatitis B or non-A, non-B viruses in human blood plasma and to the resultant products. In particular, this invention relates to the sterilization of blood plasma to render it virtually free of active hepatitis viruses, such that the valuable proteins present therein, such as factor VIII are not appreciably denatured.
2. Discussion of the Prior Art
Numerous attempts have been made to inactivate viruses such as hepatitus B virus (HBV) in mammalian, especially human blood plasma. It is the practice in some countries to effect inactivation of the hepatitis B virus in the blood plasma by contacting the plasma with a viral inactivating agent of the type which cross-links with the proteinaceous portion of hepatitis B virus, or which interacts with the nucleic acid of the virus. For instance, it is known to attempt to inactivate hepatitis B virus by contact with an aldehyde such as formaldehyde whereby crosslinking to the protein is effected and the hepatitis B virus is inactivated. It is also known to effect inactivation of the virus by contact with .beta.-propiolactone (BPL), an agent which acts on the nucleic acid of the virus. It is further known to use ultra violet light, especially after a .beta.-propiolactone treatment.
Unfortunately, these agents have only a limited ability to inactivate the viruses, and also have a deleterious effect upon other valuable protein components of the plasma. For instance, in such inactivation procedures, factor VIII is inactivated or denatured to the extent of 50-90% or more of the factor VIII present in the untreated plasma. Because of the denaturing effects of these virus inactivating agents, it is necessary in the preparation of derivatives for administration to patients to concentrate large quantities of plasma so that the material to be administered to the patient once again has a sufficient concentration of the undenatured protein for effective therapeutic treatment. This concentration, however, does not affect reduction of the amount of denatured protein. As a result, the patient not only receives the undenatured protein but a quantity of denatured protein often many times that of the undenatured protein.
For instance, if in the inactivation of hepatitis B virus in human blood plasma by .beta.-propiolactone, there is obtained as a result thereof, a plasma whose factor VIII has been 75% inactivated, the remaining 25% of the factor VIII is therefore present in such a small concentration, as a function of the plasma itself, that it is necessary to concentrate large quantities of the factor VIII to provide sufficient concentration to be of therapeutic value. Since such separation techniques do not efficiently remove denatured factor VIII from undenatured factor VIII, the material administered to the patient may contain more denatured protein than undenatured protein. Obviously, such inactivation is valuable from a standpoint of diminishing the risk of hepatitis B virus infection, however it requires the processing of large quantities of plasma and represents significant loss of valuable protein components. Furthermore, administration of large amounts of denatured proteins may render these antigenic to the host and thus give rise to autoimmune diseases, e.g. rheumatoid arthritis, or antibody to the denatured factor VIII itself.
The loss of these valuable protein components is not limited to factor VIII, the most labile of the valuable proteins in mammalian blood plasma. Similar protein denaturation is experienced in respect of the following other valuable plasma components: Coagulation factors II, VII, IX, X, Plasmin Fibrinogen, IgM, etc.
Factor VIII, however, is denatured to a larger extent that the other valuable proteins present in blood plasma.
As a result of the foregoing, except in the processing of serum albumin and stable plasma protein solution which can withstand pasteurization, it is currently the practice in the United States in respect of the processing of blood plasma and its derivatives to take no step in respect of the sterilization of the plasma for inactivation of the hepatitis viruses. As a result, recipients of factor VIII, gamma globulin, factor IX, Fibrinogen etc., must accept the risk that the valuable protein components being administered may be contaminated with hepatitis viruses. As a result, these recipients face the danger of becoming infected by these viruses and having to endure the damage which the virus causes to the liver and consequent incapacitation and illness, which may lead to death.
The BPL/UV inactivation procedure discussed above has not so far been adopted in the United States for numerous reasons, one of which lies in the fact that many researchers believe that BPL is itself deleterious since it cannot be removed completely following the inactivation and thus may remain in plasma and plasma derivatives in more than negligible amounts. BPL has been shown to be carcinogenic in animals.
Other methods for the inactivation of hepatitis B virus in the plasma are known but are usually impractical. One method involves the addition of antibodies to the plasma whereby an immune complex is formed. The expense of antibody formation and purification add significantly to the cost of the plasma production; furthermore, there is no assurance that a sufficient quantity of hepatitis B or non-A, non-B virus is inactivated. There is currently no test for non-A, non-B antibodies (although there is a test for the virus), hence, it is not possible to select plasma containing high titers of anti non-A,non-B antibody.
It is to be understood that the problems of inactivation of hepatitis viruses in plasma are distinct from the problems of inactivation of the viruses themselves due to the copresence of the desirable proteinaceous components of the plasma. Thus, while it is known how to inactivate the hepatitis B virus, crosslinking agents, e.g. glutaraldehyde, nucleic acid reacting chemicals e.g: BPL or formaldehyde, or oxidizing agents e.g. chlorox etc, it has been believed that these methods are not suitable for the inactivation of the virus in plasma due to the observation that most of these inactivating agents (sodium hypochlorite, formaldehyde, .beta.-propiolactone) denature the valuable proteinaceous components of the plasma.
It, therefore, became desirable to provide a process for the sterilization of mammalian blood plasma which does not substantially denature the valuable components of the plasma and which does not entail the use of a potentially carcinogenic agent. More especially, it is desirable to provide blood plasma in which all of the hepatitis viruses present are inactivated and in which denatured protein such as factor VIII account for only a small amount of the total amount of these proteins in the plasma.
A further advantage of the proposed procedures is the fact that plasma, or plasma protein solutions so treated become totally clear and transparent as a result of the removal of plasma lipids. Furthermore, the clarity is maintained indefinitely on storage at 4.degree. C. This has important advantages over untreated plasma or plasma protein solutions in that:
(1) it becomes easy to detect bacterial contamination by inspection, a procedure which is difficult in turbid suspensions; and
(2) development of microaggregates of precipitated lipoproteins which occurs normally on cold storage of untreated plasma or plasma protein solutions, is prevented, thus avoiding the potentially adverse effects of infusion of such microaggregates which may lodge in pulmonary, renal, or cerebral capillaries and obstruct them.
Finally, the proposed procedures permit the inactivation of viral infectivity in source plasma from chronic carriers of hepatitis B virus used for preparation of HBV viral vaccines, permitting a safer manufacturing process and a safer product. The rationale and concepts described above are illustrated in FIG. 1.